Cellular Characteristics Of Sertoli Cell Within The Testis Of Chinese Soft-shelled Turtle,Pelldiscus Sinensis

Spermatogenesis is a complex process required of all vertebrate males in which progenitor cells mitotically divide,undergo meiosis,and differentiate into motile spermatozoa within the testes.The testis parenchyma consists of numerous seminiferous tubules and interstitial space.Each seminiferous tubule is consists of developing germ cells and only epithelial cell known as Sertoli cell or nurse cell.Sertoli cell play a central role in the regulation of spermatogenesis and the production of male germ cells.Understanding interactions between Sertoli cells and germ cells is critical for discerning testicular organization and physiologic relationships between Sertoli cells and germ cells.Understanding these cellular relationships in lower vertebrates may help unravel the evolution and development of Sertoli cell-germ cell interrelationships among higher vertebrates.Unlike mammals,there have been few studies on the cytology of the Sertoli cell in non-mammalian species.The Chinese soft-shelled turtle is an ancient type of reptile found in China and other several Asiatic countries.It has a great importance in aquaculture and as a food item,and is,of significant economic and pharmacological value.Though a little information available on the structure and function of the Sertoli cell within the testis of class Reptilia,much of what is known is incomplete.The junctional complex between adjacent Sertoli cells and,Sertoli cells with germ cells have been observed in a few species of reptiles,but no comprehensive data available that describes their structure within the seminiferous epithelium of turtles.Furthermore,some aspects of testicular function have yet to be explored within reptiles and even in mammals.It is well known that Sertoli cell undergoes lipid cycle in the seasonally breeding animals;however,the utilization of lipid droplets within Sertoli cells during spermatogenesis is still largely unknown.Sertoli cell can phagocytosis the residual bodies and or residual spermatozoa after spermiation,however,this mechanism still remain mysterious.The current study clearly demonstrate the seasonal effects on morphological changes in Sertoli cells,with a focus on the functional aspects of Sertoli cells in relation to the developing germ cells and provide a detailed analysis of germ cell development strategy during spermatogenesis of Chinese soft-shelled turtle.Furthermore,we explore the first time a novel role of lipophagy in the consumption of lipid droplets within Sertoli cells in the testes.We also have investigated the characterization of inter-Sertoli cell tight junction and gap junction during spermatogenesis.Finally,current study demonstrated the novel cellular mechanism of entosis along with evidence of autophagy within the Sertoli cells for eliminating the residual spermatozoa in the seminiferous tubules during hibernation period,which is an essential program for the next reproductive cycle of the Chinese soft-shelled turtle.Thus,the current study has provided the comprehensive knowledge about the ultrastructre of Sertoli cell and meantime has given a new perspective of Sertoli cell functionality on germ cells.However,this study provides novel evidence of lipophagy and entosis within the Sertoli cell at cellular level for the first time.Further studies will be required to explore these mechanisms in detail at molecular level or develops in vitro models.Experiment-1 Cytological study on Sertoli cells and their interactionswith germ cells during annual reproductive cycle in turtleSertoli cells(SCs)play a central role in the development of germ cells within functional testes and exhibit varying morphology during spermatogenesis.The present study investigated the seasonal morphological changes in SCs in the reproductive cycle of Pelodiscus sinensis by light microscopy,transmission electron microscopy and immunohistochemistry.During hibernation period with the quiescent of spermatogenesis,several autophagosomes were observed inside the SCs,the processes of which retracted.In early spermatogenesis,when the germ cells started to proliferate,the SCs contained numerous lipid droplets instead of autophagosomes.In late spermatogenesis,the SCs processes became very thin and contacted several round/elongated spermatids in pockets.At this time,abundant endoplasmic reticulum and numerous mitochondria were present in the SCs.The organization of the tight junctions and the adherens junctions between the SCs and germ cells also changed during the reproductive cycle.Moreover,SCs were involved in the formation of cytoplasmic bridges,phagophores and exosome secretions during spermatogenesis.Tubulobulbar complexes(TBC)were also developed by SCs around the nucleus of the spermatid at the time of spermiation.Strong,positive expression of vimentin was noted on the SCs during late spermatogenesis compared with the hibernation stage and the early stage of spermatogenesis.These data provide clear cytological evidence about the seasonal changes in SCs,corresponding with their different roles in germ cells within the Chinese soft-shelled turtle,Pelodiscus sinensis.Experiment-2 Characterization of Inter-Sertoli cell tight junctions andgap junctions in the testis of Chinese soft-shelled turtleIt is conceivable that early developing germ cells must across the basal to the luminal region of seminiferous tubules(STs)during spermatogenesis is associated with extensive restructuring of junctional complex.However,very limited information is documented about these junctional complexes in reptiles.In the present study we have determined the localization of inter-Sertoli cell tight junctions(TJ's)protein CLDN11 and gap junction protein Cx43 in the testis of Pelodiscus sinensis during spermatogenesis.In early spermatogenesis,weak immunoreactivity of CLDN11 and focal localization of Cx43 was observed around the Sertoli cell in the luminal region,but completely delaminating from the basal compartment of STs.In late spermatogenesis,strong focal to linear localization of CLDN11and Cx43 was detected at the points of contact between two Sertoli cells and around the early stages of primary spermatocytes in the basal compartment of STs.In late spermatogenesis,localization of CLDN11and Cx43 was drastically reduced and seen only around Sertoli cells and spermatogonia near the basal membrane.However,transmission electron microscopy revealed that the inter-Sertoli cell tight junctions were present within the basal compartment of STs during mid spermatogenesis.Gap junctions were observed between Sertoli cells,and Sertoli cells with spermatogonia and primary spermatocytes throughout spermatogenesis.Moreover,adherens and hemidesmosomes junctions were observed during spermatogenesis.The above findings collectively suggest that the intensity and localization of TJ's and gap junctions varies according to the spermatogenetic stages,this might be correlated with germ cell differentiation during spermatogenesis.Experiment-3 Novel cellular evidence of lipophagy within the Sertoli cells during spermatogenesis in the turtleSpermatogenesis is a complex process producing haploid spermatozoa,and the formation of lipid droplets(LDs)within Sertoli cells is critical to maintaining normal spermatogenesis.However,the utilization of LDs within Sertoli cells is still largely unknown.In the present study,proliferation of spermatogonial cells had begun in May,whereas the meiotic cells occurred predominately in July and majority of spermiogenic cells were observed in the seminiferous tubules in October.However,TEM and Oil Red O staining demonstrated that a larger number of LDs had accumulated within the Sertoli cells in May compared to that in October.There were several LDs attached to the isolation membrane/phagophore,suggesting that the LDs may be a source of endogenous energy for the biogenesis of autophagosomes.The LDs were enclosed within the autophagosomes in May,whereas,autophagosomes and mitochondria were directly attached with large LDs within the Sertoli cells in October.Furthermore,immunohistochemistry results demonstrated the stronger localization of LC3 on the Sertoli cells in May than in October.This study is the first to provide clear evidence of the two different modes of lipophagy for lipid consumption within Sertoli cells,which is a key aspect of Sertoli germ cell communication during spermatogenesis.Experiment-4 Entosis acts as a novel way within Sertoli cells to eliminate spermatozoa in seminiferous tubuleThe present study was designed to investigate the hypothesis that in vivo entosis is a novel pathway for eliminating spermatozoa in the seminiferous tubules(ST)during hibernation of the Chinese soft-shelled turtle.Western blot analysis revealed that the expression of LAMP1 in the testis was significantly higher during hibernation than that during non-hibernation.Immunohistochemistry reaction showed that LAMP1-positive substance was distributed within the Sertoli cells of the testis.Further examination by transmission electron microscopy(TEM),many degraded spermatozoa being enwrapped within large entotic vacuoles in Sertoli cells.The nucleus and the flagellum of the spermatozoa were shown to be decomposed and digested inside entotic vacuoles within Sertoli cells.More than two spermatozoa heads were always observed in each internalized vacuoles.Deserving note is that,a number of different autophagosomes,including initial autophagic vesicles and degradative autophagic vesicles were found inside the entotic vacuoles of the Sertoli cells during hibernation.At the end of hibernation,entotic vacuoles and their autophagosomes disappeared,and numerous large lipid droplets(LDs)appeared within the Sertoli cells.Adherens junctions were apparent between Sertoli cells and developing germ cells,which is the ultrastructural basis of entosis.Taken together,the results presented here show that in the turtle:1)entosis with internal autophagosomes can take place within normal body cells during hibernation;2)spermatozoa,as a highly differentiated cell can be internalized and degraded within Sertoli cell by entosis in vivo,which is in favor of the next reproductive cycle in the turtle.